Not applicable.
The invention provides isolated nucleic acid and amino acid sequences of BK beta subunits 2, 3, and 4, which are auxiliary subunits of Slo potassium channels, antibodies to BK beta subunits 2-4, methods of detecting BK beta subunits 2-4, methods of screening for modulators of Slo potassium channels comprising BK beta subunits 2, 3, or 4, and kits for screening for modulators of Slo family potassium channels comprising BK beta subunits 2, 3, or 4.
Potassium channels are involved in a number of physiological processes, including regulation of heartbeat, dilation of arteries, release of insulin, excitability of nerve cells, and regulation of renal electrolyte transport. Potassium channels are thus found in a wide variety of animal cells such as nervous, muscular, glandular, immune, reproductive, and epithelial tissue. These channels allow the flow of potassium in and/or out of the cell under certain conditions. For example, the outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, second messengers, extracellular ligands, and ATP-sensitivity.
Potassium channels are made by alpha subunits that fall into 8 families, based on predicted structural and functional similarities (Wei et al., Neuropharmacology 35(7):805-829 (1997)). Three of these families (Kv, eag-related, and KQT) share a common motif of six transmembrane domains and are primarily gated by voltage. Two other families, CNG and SK/IK, also contain this motif but are gated by cyclic nucleotides and calcium, respectively. The three other families of potassium channel alpha subunits have distinct patterns of transmembrane domains. Slo family potassium channels, or BK channels have seven transmembrane domains (Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94(25):14066-71 (1997)) and are gated by both voltage and calcium or pH (Schreiber et al., J. Biol. Chem. 273:3509-16 (1998)). Another family, the inward rectifier potassium channels (Kir), belong to a structural family containing 2 transmembrane domains, and an eighth functionally diverse family (TP, or xe2x80x9ctwo-porexe2x80x9d) contains 2 tandem repeats of this inward rectifier motif.
Potassium channels are typically formed by four alpha subunits, and can be homomeric (made of identical alpha subunits) or heteromeric (made of two or more distinct types of alpha subunits). In addition, potassium channels made from Kv, KQT and Slo or BK subunits have often been found to contain additional, structurally distinct auxiliary, or beta, subunits. These subunits do not form potassium channels themselves, but instead they act as auxiliary subunits to modify the functional properties of channels formed by alpha subunits. For example, the Kv beta subunits are cytoplasmic and are known to increase the surface expression of Kv channels and/or modify inactivation kinetics of the channel (Heinemann et al., J. Physiol. 493:625-633 (1996); Shi et al., Neuron 16(4):843-852 (1996)). In another example, the KQT family beta subunit, minK, primarily changes activation kinetics (Sanguinetti et al., Nature 384:80-83 (1996)).
Slo or BK potassium channels are large conductance potassium channels found in a wide variety of tissues, both in the central nervous system and periphery. They play a key role in the regulation of processes such as neuronal integration, muscular contraction and hormone secretion. They may also be involved in processes such as lymphocyte differentiation and cell proliferation, spermatocyte differentiation and sperm motility. Three alpha subunits of the Slo family have been cloned, i.e., Slo1, Slo2, and Slo3 (Butler et al., Science 261:221-224 (1993); Schreiber et al., J. Biol. Chem., 273:3509-16 (1998); and Joiner et al., Nature Neurosci. 1: 462-469 (1998)). These Slo family members have been shown to be voltage and/or calcium gated, and/or regulated by intracellular pH.
A BK (or Slo) beta subunit that associates with Slo/BK potassium channels has been cloned and called BK beta 1 (Dworetzky et al., J. Neurosci. 16:4543-50 (1996); U.S. Pat. No. 5,776,734; see also Xia et al., J. Neurosci. 19:5255-5264 (1999), Wallner et al., Proc. Nat""l Acad. Sci. USA 96:4137-4142 (1999), Ali Riazi et al., Genomics 62:90-94 (1999), and EP 0 936 271 A1). BK beta 1 has short cytoplasmic N and C termini and has two membrane-spanning regions with a large extracellular loop. Functionally, BK beta 1 modulates the activation kinetics and calcium sensitivity of Slo1 channels (McManus et al., Neuron 14:645-50 (1995)). BK beta 1 also increases calcium sensitivity, sensitivity to extracellular toxins, and decreases the activation rate for Slo1 channels.
Additional beta subunits for Slo family potassium channels remain to be identified. The discovery and characterization of those Slo or BK beta subunits will provide important insights into how Slo potassium channels function in different environments, and how they respond to various activation mechanisms. Such information would also allow the identification of modulators of Slo potassium channels and the use of such modulators as therapeutic agents.
The present invention thus identifies for the first time BK beta 2, BK beta 3, and BK beta 4, each of which is a beta subunit of a Slo family potassium channel. These BK beta subunits have neither been previously cloned nor identified, and the present invention provides both the amino acid and nucleotide sequences of BK beta 2, BK beta 3, and BK beta 4.
In one aspect, the present invention provides an isolated nucleic acid encoding a beta subunit of a potassium channel, wherein the beta subunit: (i) forms, with at least one alpha unit, a Slo potassium channel; (ii) comprises an amino acid sequence that has greater than about 70% identity to the S1-S2 region of BK beta 2, BK beta 3, or BK beta 4; and (iii) specifically binds to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO:5.
In one aspect, the present invention provides an isolated nucleic acid encoding a beta subunit of a potassium channel, wherein the beta subunit: (i) forms, with at least one alpha subunit, a Slo potassium channel; (ii) comprises an amino acid sequence that has greater than about 70% identity to the S1-S2 region of BK beta 2, BK beta 3, or BK beta 4; and (iii) specifically binds to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; wherein said nucleic acid either: (i) selectively hybridizes under moderately stringent hybridization conditions to a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; or (ii) encodes a protein which could be encoded by a nucleic acid that selectively hybridizes under moderately stringent hybridization conditions to a nucleotide of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
In one embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as primers selected from the group consisting of:
5-ATGACAGCCTTTCCTGCCTCAGGGAAG-3 (SEQ ID NO:7)
5-AGATTTCTCTGCTCTTCCTTTGCTCCTCC-3 (SEQ ID NO:8)
5-GGCTGGCTGGACTGTAGAAGCATG-3 (SEQ ID NO:9)
5-GAGGCTGTCCAGATAAATCCCAAGTGC-3 (SEQ ID NO:10)
5-GGACTGAGAAGCCCATCATGGCAAACC-3 (SEQ ID NO:11);
5-ATGGCGAAGCTCCGGGTGGCTTAC-3 (SEQ ID NO:12)
5-TTAAGAGAACTTGCGCTTCTTCATGG-3 (SEQ ID NO:13)
5-GATGTGCTTCTGCATCGCACTCATG-3 (SEQ ID NO:14); and
5-AAGATGTCGATATGGACCAGTGGCC-3 (SEQ ID NO:15)
5-TTATCTATTGATCCGTTGGATCCTCTC-3 (SEQ ID NO:16)
5-CTCCTTCAGCTGTCCTCCAGACTGC-3 (SEQ ID NO:17)
5-GTCCCAGTAGAATAGCTCGGTCCTC-3 (SEQ ID NO:18).
In one embodiment, the nucleic acid encodes a beta subunit having a molecular weight of about between 24-34 kDa, 18-28 kDa, or 22-32 kDa. In another embodiment, the nucleic acid encodes human BK beta 2, 3, or 4. In another embodiment, the nucleic acid encodes SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
In another aspect, the present invention provides an isolated beta subunit of a potassium channel, wherein the beta subunit: (i) forms, with at least one alpha subunit, a Slo potassium channel; (ii) comprises an amino acid sequence that has greater than about 70% identity to the S1-S2 region of BK beta 2, BK beta 3, or BK beta 4; and (iii) specifically binds to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
In one embodiment, the beta subunit is human BK beta 2, 3, or 4. In another embodiment, the beta subunit has the sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
In another aspect, the present invention provides an antibody that selectively binds to a beta subunit as described above.
In another aspect, the present invention provides an expression vector comprising a nucleic acid as described above.
In another aspect, the present invention provides host cell transfected with an expression vector as described above.
In another aspect, the present invention provides a method for identifying a compound that increases or decreases ion flux through a potassium channel, the method comprising the steps of: (i) contacting the compound with a eukaryotic host cell or cell membrane in which has been expressed potassium channel comprising a beta subunit, wherein the beta subunit:(a) forms, with at least one alpha subunit, a Slo potassium channel; (b) comprises an amino acid sequence that has greater than about 70% identity to the S1-S2 region of a BK beta 2, BK beta 3, or a BK beta 4; and (c) specifically binds to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; and (ii) determining the functional effect of the compound upon the cell or cell membrane expressing the Slo potassium channel.
In one embodiment, the functional effect is determined by measuring changes in current or voltage. In another embodiment, the beta subunit is recombinant. In one embodiment, the beta subunit is human BK beta 2, 3, or 4. In another embodiment, the beta subunit has the sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
In another aspect, the present invention provides a method of detecting the presence of BK beta 2, 3 or 4 in a sample, the method comprising the steps of: (i) isolating a biological sample; (ii) contacting the biological sample with a BK beta 2, 3, or 4-specific reagent that selectively associates with BK beta 2, 3, or 4; and, (iii) detecting the level of BK beta 2, 3, or 4-specific reagent that selectively associates with the sample.
In one embodiment, the BK beta specific reagent is selected from the group consisting of: BK beta specific antibodies, BK beta specific oligonucleotide primers, and BK beta specific-nucleic acid probes. In another embodiment, the sample is from a human.
In another aspect, the present invention provides, in a computer system, a method of screening for mutations of human BK beta 2, BK beta 3, or BK beta 4 genes, the method comprising the steps of: (i) entering into the system at least about 25 nucleotides of first nucleic acid sequence encoding a beta subunit having a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; (ii) comparing the first nucleic acid sequence with a second nucleic acid sequence having substantial identity to the first nucleic acid sequence; and (iii) identifying nucleotide differences between the first and second nucleic acid sequences.
In one embodiment, the second nucleic acid sequence is associated with a disease state.
In aspect, the present invention provides a computer readable substrate comprising the first nucleic acid sequence.
In another aspect, the present invention provides, in a computer system, a method for identifying a three-dimensional structure of BK beta 2, BK beta 3, or BK beta 4 subunits, the method comprising the steps of: (i) entering into the system an amino acid sequence of at least 25 amino acids of a beta subunit or at least 75 nucleotides of a gene encoding the beta subunit, the beta subunit having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; and (ii) generating a three-dimensional structure of the beta subunit encoded by the amino acid sequence.
In another aspect, the present invention provides a computer readable substrate comprising the three dimensional structure of the beta subunit.
In one embodiment, the amino acid sequence is a primary structure and wherein said generating step includes the steps of: (i) forming a secondary structure from said primary structure using energy terms determined by the primary structure; and (ii) forming a tertiary structure from said secondary structure using energy terms determined by said secondary structure. In another embodiment, said generating step further includes the step of forming a quaternary structure from said tertiary structure using anisotropic terms encoded by the tertiary structure. In another embodiment, the method further comprises the step of identifying regions of the three-dimensional structure of a BK beta 2, BK beta 3 or BK beta 4 subunit that bind to ligands and using the regions to identify ligands that bind to the beta subunit.